REVERSIBLE PHOSPHORYLATION AS A CONTROLLING FACTOR FOR SUSTAINING CALCIUM OSCILLATIONS IN HELA-CELLS - INVOLVEMENT OF CALMODULIN-DEPENDENT KINASE-II AND A CALYCULIN A-INHIBITABLE PHOSPHATASE

The role of reversible phosphorylation in histamine-induced Ca2+ oscil lations in HeLa cells has been investigated by using various activator s and inhibitors of protein kinases and phosphatases. Electroporation was employed to introduce impermeable materials into single cells, whi ch proved to be a useful and convenient tool, Of the kinases examined, cAMP-dependent kinase, protein kinase C, and calmodulin-dependent kin ase II (CaMK II), only CaMK II was essential. When added during oscill ations, both W-7, a calmodulin antagonist, and KN-62, a specific CaMK II inhibitor, caused one large Ca2+ spike before halting the process. Introduction of the Ca2+/calmodulin-independent catalytic domain of Ca MK II into the cells forestalled their response to histamine. These re sults show that intracellular Ca2+ cannot oscillate when CaMK II is lo cked in either the inactive or the stimulated state. External Ca2+ ele ctroporated into cells preloaded with the catalytic domains was quickl y removed (but not when the cells were pretreated with the endoplasmic reticulum Ca2+-ATPase inhibitor, tapsigargin), indicating that the AT P-driven Ca2+ pump was somehow activated by CaMK II. Protein phosphata se inhibitors calyculin A and okadaic acid abolished ongoing oscillati ons and, when added at low concentrations, prolonged the interspike in terval. Immunoprecipitation experiments with P-32(i)-labeled cells pro vided the first evidence that inositol 1,4,5-trisphosphate receptor (I P(3)R) was phosphorylated by CaMK II in vivo. The extent of phosphoryl ation was increased in the presence of histamine, significantly enhanc ed by calyculin A, and greatly reduced by W-7. Our observations are co nsistent with the concept that repetitive phosphorylation-dephosphoryl ation cycles regulating IP(3)R and Ca2+ pumps are a controlling factor for sustained Ca2+ oscillations in HeLa, and possibly other, cells.